Pyrosequencing-based analysis of bacterial community and metabolites profiles in Korean traditional seafood fermentation: a flatfish-fermented seafood

Bacterial community and metabolites were analyzed in a flatfish jeotgal, a Korean fermented seafood. Inverse relationship of pH and 16S rRNA gene copy number was identified during fermentation. Lactobacillus was the predominant bacterial genus. Increase of Firmicutes was a common characteristic shared by other fermented seafood. Fructose, glucose, and maltose were the major metabolites.     

Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 μM. PMA at a concentration of 50 μM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration

Aims: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. Methods and Results: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). Moreover, species‐specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species‐specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. Conclusions: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. Significance and Impact of the Study: This study provides important information that may be used to extend the shelf life of kava beverages.     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

PMA结合ddPCR检测食品中金黄色葡萄球菌的研究

研究了将叠氮溴化丙锭(PMA)与微滴式数字PCR(ddPCR)技术相结合,用于金黄色葡萄球菌活菌的检测。结果表明,强烈光照15 min,可以使PMA与死菌DNA共价交联,同时钝化游离的PMA;可以有效抑制金黄色葡萄球菌死菌DNAPCR扩增的PMA终浓度为2.0μg/m L;不抑制活菌DNA扩增的PMA最高浓度是5.0μg/m L。在不同死、活菌比例下,PMA-ddPCR可以定量检测活菌,避免了死菌DNA的干扰,本方法的检出限为10 copy/20μL。利用PMA-ddPCR检测人工污染鸡肉样品,最低可检出102cfu/m L的金黄色葡萄球菌。表明PMA-ddPCR方法的灵敏度高。     

干湿交替水分胁迫对水稻土细菌群落影响的研究

[目的]连续3次风干-湿润循环培养水稻土,在DNA和RNA水平下,探究细菌对干湿交替胁迫的响应机制,明确风干水稻土能否代替新鲜土壤进行细菌群落组成分析.[方法]针对我国江苏省常熟市水稻土,开展新鲜土壤的3次风干-湿润循环连续培养处理(每次循环中风干、湿润状态各维持7 d),在DNA和RNA水平应用16S rRNA基因高...     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.     

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets

AIMS: To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. METHODS AND RESULTS: Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. CONCLUSIONS: Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake

The fate of antibiotic resistance genes (ARGs) in environmental microbial communities is of primary concern as prodromal of a potential transfer to pathogenic bacteria. Although of diverse origin, the persistence of ARGs in aquatic environments is highly influenced by anthropic activities, allowing potential control actions in well‐studied environments. However, knowledge of abundance and space–time distribution of ARGs in ecosystems is still scarce. Using quantitative real‐time PCR, we investigated the presence and the abundance of twelve ARGs (against tetracyclines, β‐lactams, aminoglycosides, quinolones and sulphonamides) at different sampling sites, depths and seasons, in Lake Maggiore, a large subalpine lake, and in the area of its watershed. We then evaluated the correlation between each ARG and a number of ecological parameters in the water column in the deepest part of the lake. Our results suggest the constitutive presence of at least four ARGs within the bacterial community with a high proportion of bacteria potentially resistant to tetracyclines and sulphonamides. The presence of these ARGs was independent of the total bacterial density and temperature. The dynamics of tet(A) and sulII genes were, however, positively correlated with dissolved oxygen and negatively to chlorophyll a, suggesting that the resistant microbes inhabit specific niches. These observations indicate that the lake is a reservoir of antibiotic resistances, highlighting the need of a deeper understanding of the sources of ARGs and the factors allowing their persistence in waters.     

老年与中青年患者下呼吸道感染病原菌的对比分析

目的对比分析老年与中青年患者下呼吸道感染病原菌的分布规律及其耐药性特点,以指导临床合理用药。方法将研究对象分为老年(≥60岁)和中青年(20～59岁)2组;采用API系统进行菌种鉴定;采用K-B法进行药敏试验;采用纸片扩散表型确证法进行超广谱β-内酰胺酶(ESBLs)测定;采用SPSS 13.0进行χ2检验。结果老年组的真菌分离率显著高于中青年组,以白色假丝酵母菌最多;中青年组的G-杆菌分离率显著高于老年组,以铜绿假单胞菌最多;老年组主要致病菌对多数药物的耐药率比中青年组有增高趋势,但差异多无统计学意义;老年组肺炎克雷伯菌的ESBLs阳性率显著高于中青年组。结论老年与中青年患者下呼吸道感染的病原菌分布及耐药性存在一定差异。     

The Relationship between Local Abundance and Distribution of Rain Forest Trees across Environmental Gradients in India

We tested whether local abundance of rain forest trees in the medium elevation wet forests of the southern Western Ghats (WG) was related to environmental tolerance, life form, and geographical range. We selected trees in medium elevation wet forests (750–1700 m asl) of the southern WG, using two data bases: a small plot (30 × 30 m) data base of 288 species of trees (≥ 3 cm dbh) in 33 plots totaling 2.97 ha, and a data base of 135 species of tree (≥ 10 cm dbh) in larger plots of 1 ha each, totaling 4.84 ha. The species density per hectare and number of records in the plot network was used in a factor analysis to give a measure of the local abundance of each species. The altitude and seasonality ranges of these species in the WG was assessed from independent data bases and used to generate an environmental tolerance score. Results indicated that as a species became locally more abundant, it occurred across a wider range of environmental gradients, but regional distribution was not related to geographical distribution. Understory species tended to be rarer with smaller range sizes and lower environmental tolerances than overstory species. Climate change is predicted to have drastic effects on restricted range species with limited environmental tolerances.     

High-throughput nucleotide sequence analysis of diverse bacterial communities in leachates of decomposing pig carcasses

The leachate generated by the decomposition of animal carcass has been implicated as an environmental contaminant surrounding the burial site. High-throughput nucleotide sequencing was conducted to investigate the bacterial communities in leachates from the decomposition of pig carcasses. We acquired 51,230 reads from six different samples (1, 2, 3, 4, 6 and 14 week-old carcasses) and found that sequences representing the phylum Firmicutes predominated. The diversity of bacterial 16S rRNA gene sequences in the leachate was the highest at 6 weeks, in contrast to those at 2 and 14 weeks. The relative abundance of Firmicutes was reduced, while the proportion of Bacteroidetes and Proteobacteria increased from 3–6 weeks. The representation of phyla was restored after 14 weeks. However, the community structures between the samples taken at 1–2 and 14 weeks differed at the bacterial classification level. The trend in pH was similar to the changes seen in bacterial communities, indicating that the pH of the leachate could be related to the shift in the microbial community. The results indicate that the composition of bacterial communities in leachates of decomposing pig carcasses shifted continuously during the study period and might be influenced by the burial site.     

基于实时定量PCR技术的育龄期细菌性阴道病患者阴道菌群结构分析及其临床应用

目的 探讨育龄期女性细菌性阴道病（bacterial vaginosis,BV）患者阴道优势菌群变化及其与临床指标的相关性。方法 选择本院门诊确诊的育龄期BV患者35例和同期入院体检的健康体检女性37例,无菌拭子采集阴道中段壁分泌物,提取细菌基因组DNA,采用实时定量PCR技术（real-time quantitative PCR,qPCR）进行阴道优势细菌检测,并进行其与临床指标如阴道pH和Nugent评分相关性分析。结果 乳杆菌属细菌及其种水平的细菌如惰性乳杆菌、卷曲乳杆菌、詹氏乳杆菌等在BV患者中均显著下降,BV相关阴道致病细菌如加德纳菌属、奇异菌属、埃格特菌属、巨型球菌I型菌属、纤毛菌属和普氏菌属显著升高（P<0.05）。阴道致病菌群与阴道pH和Nugent评分呈显著正相关,而阴道卷曲乳杆菌和惰性乳杆菌与其呈负相关。结论 育龄期BV患者阴道优势菌群显著失衡,并与阴道pH和Nugent评分显著相关,提示阴道优势菌群改变参与BV发生发展。